Multivariate analysis of the phytochemical composition and antioxidant properties in twenty-five accessions across three Achillea species

This study explored the chemical composition, antioxidant activity, and total phenol content of aerial parts from 25 accessions of three Achillea species (Achillea wilhelmsii C. Koch, Achillea vermicularis Trin., and Achillea tenuifolia Lam.). The plants were collected from various natural habitats across Iran, encompassing regions such as Central, Western, Southern, Northern, Western, and Northwestern parts of the country. Subsequently, they were grown together under field conditions. The study revealed significant variation in essential oil yields among accessions of A. wilhelmsii, ranging from 0.01 to 0.107%, A. vermicularis with a range of 0.075 to 1.5%, and A. tenuifolia showing a variation of 0.1 to 2%. The study utilized Gas Chromatography–Mass Spectrometry (GC–MS) analysis, revealing 75, 49, and 75 compounds in the essential oils of A. wilhelmsii, A. tenuifolia, and A. vermicularis, respectively. Major components included camphor, 1,8-cineole, anethole, α-pinene, and phytol in A. wilhelmsii, 1,8-cineole, camphor, levo-carvone, and δ-terpinene in A. vermicularis, and β-cubebene, elixene, β-sesquiphellandrene, 1,8-cineole, camphor, and δ-terpinene in A. tenuifolia. The essential oil compositions of A. wilhelmsii and A. vermicularis were predominantly characterized by oxygenated monoterpenes, whereas that of A. tenuifolia was characterized by sesquiterpenes. Cluster analysis grouped accessions into three clusters, with A. tenuifolia forming a distinct group. Principal Component Analysis (PCA) triplot (62.21% of total variance) confirmed these results and provided insights into compound contributions. Furthermore, total phenolic content and antioxidant activity of the accessions of three species were assessed over 2 years. A. tenuifolia exhibited the highest levels in both categories, with statistically significant linear regression between antioxidant activity and total phenol content for A. tenuifolia and A. wilhelmsii. These findings emphasize significant phytochemical diversity within Achillea species, positioning them as promising natural sources of antioxidants. Further exploration and selection of specific accessions within each species are crucial for unlocking their medicinal potential and supporting cultivation and conservation efforts.


Phytochemicals composition of the essential oils
GC-MS analysis was performed using a Varian CP-3800 instrument equipped with a VF-5 capillary column (30 m × 0.25 mm i.d., film thickness 0.25 µm).Helium was used as the carrier gas at a flow rate of 1 mL/min, and the temperature program was set at 60 °C for 1 min, followed by an increase to 250 °C at a rate of 3 °C/min, and held for 10 min.The injector and detector temperatures were maintained at 250 °C and 280 °C, respectively.To identify the components of the essential oils, the retention index (RI) was utilized by subjecting n-alkanes (C6-C24) to programmed temperature conditions.The resulting RI values were then compared to the internal reference MS library (Wiley 7.0) and published data in the literature 36 .www.nature.com/scientificreports/

Extracting the plant extracts
The plant materials were dried at room temperature to remove moisture.Once fully dried, the plants were ground into a fine powder using a mill.For extraction, 5 g of each powdered sample was accurately weighed and transferred to separate Erlenmeyer flasks.To each flask, 50 mL of 80% methanol solvent was added.Extraction was carried out using maceration, where the plant powder was soaked and agitated in the methanol.A magnetic stir plate and orbital shaker set to 150 rpm were used to gently mix the samples at 25 °C for 24 h.After maceration, the mixtures were strained through filter paper to separate the extracts from insoluble residues.The filtered extracts were concentrated by evaporating the methanol under reduced pressure using a rotary evaporator.Then, the pure extract was collected in a small container and stored at 4 °C until total phenol and antioxidant activity analyses.Prior to the analyses, the samples were dried and used immediately.

Total phenolic content (TPC)
One millilitre of diluted extract (0.1 g in 10 mL of distilled water) was combined with 1 ml of 6 M HCl and 5 mL of 75% methanol/water solution.The resulting mixture was subjected to shaking for 2 h at 90 °C in a water bath.Subsequently, the solution was diluted to a final volume of 10 ml using distilled water.One milliliter of this diluted solution was mixed with 5 ml of previously tenfold diluted Folin & Ciocalteau reagent and 15 ml of sodium carbonate solution (7 g/100 mL).The resulting mixture was brought to a final volume of 100 mL with distilled water.The absorbance of the solution at 760 nm was measured using a spectrophotometer, comparing it against a blank prepared using distilled water instead of the extract, which had undergone the same extraction steps.The experiment was conducted in triplicate, and our methodology closely followed the approach described by Çam et al. 37 , with the exception that we employed four different concentrations of gallic acid solution (1.0, 0.4, 1.6, and 2.2 mg per milliliter) in this study.Finally, the total phenolic content in the extract was quantified and reported as milligrams of gallic acid per milliliter of the sample extract.

Antioxidant activity
The antioxidant activity of the extract was assessed following the methodology of Brand-Williams et al. 38 , with minor modifications.The experiment employed four different concentrations including 10, 100, 250, and 500 ppm of the extract (0.1, 1, 2.5, and 5 mg in 10 mL of distilled water, respectively).Subsequently, 0.1 ml of each concentration was added to 3.9 ml of a 6 × 10 -5 mol/L methanol DPPH solution.For the control sample, 0.1 ml of methanol were mixed with 3.9 ml of the methanolic DPPH solution.The spectrophotometer was calibrated using pure methanol as the zero reference.After an incubation period of 30 min, the absorbance of all samples was measured at a wavelength of 515 nm.

Statistical analysis
The PCA analysis was conducted using version 9.1 of the Statistical Analysis Software (SAS Institute, Cary, NC) for Windows.A heat map clustering analysis was performed to visualize the similarity patterns among samples based on their phytochemicals components values.Hierarchical clustering was applied using Euclidean distance measure and the arithmetic mean method (UPGMA).The heat map displays the clustering of accessions on the y-axis and phytochemicals components on the x-axis, with color intensity indicating the standardized value for each trait in each accession.This analysis helped group accessions exhibiting similar response patterns.Also, a correlation heat map was generated to examine relationships between phytochemicals components.Pairwise correlation coefficients between the components were computed and plotted in a color-coded matrix, with red indicating positive correlation and blue representing negative correlation.The correlation coefficient values were depicted based on their absolute strengths.The results of the clustering, heat map correlation, and triplot analyses were visualized as a colored heat map using MetaboAnalyst 39 .Additionally, the graphs were created using Prism 9 (GraphPad).

Essential oil yield
The essential oil yield of 25 accessions from three Achillea species (A.wilhelmsii, A. tenuifolia, and A. vermicularis) was evaluated over two consecutive years.Figure 1 presents the essential oil yields for each accession during the first and second year of cultivation.In the first year, yields ranged from 0.01 to 1.2% whereas in the second year yields were generally higher between 0.02 and 2%.Statistically significant differences were observed between the 2 years for all three species (p < 0.01).Yields increased for the majority of accessions in the second compared to the first year.Certain A. tenuifolia accessions such as T4 exhibited notably higher essential oil production in the second year.Based on the ranges observed, A. tenuifolia accessions generally exhibited the highest essential oil yields, followed by A. vermicularis, with A. wilhelmsii having the lowest yields.In the first year, A. tenuifolia accessions produced 0.1-1.04%oil, A. vermicularis yields varied from 0.12 to 1.2%, while A. wilhelmsii yields were under 0.1%.Similarly, in the second year, A. tenuifolia accessions yielded 0.71-2%, A. vermicularis varied from 0.075 to 1.45%, and A. wilhelmsii increased but remained low at 0.02-0.107%.The results demonstrate considerable variation in the essential oil yields among the accessions and between the 2 years.In general, it can be observed that the essential oil yields tend to be higher in the second year compared to the first year for all three Achillea species.This finding suggests that the plants undergo certain physiological changes that positively influence essential oil production as they mature.One possible explanation for the increased essential oil yield in the second year is the establishment and development of the plants during the first year.As perennial plants, the first year is typically characterized by a longer growth period until flowering, which occurred in August.
The extended growth period in the first year may have prioritized vegetative growth over secondary metabolite production, resulting in lower essential oil yields.In contrast, the second year exhibited a shorter growth www.nature.com/scientificreports/cycle, with flowering occurring in May.This shorter growth period likely allowed for increased accumulation of phenolic antioxidants in the aerial parts by the time of flowering, leading to higher essential oil yields.The observed increase in essential oil yield in the second year highlights the importance of considering the stage of plant maturity when studying essential oil production in perennial species.It suggests that the developmental stage and growth cycle significantly influence the biosynthesis and accumulation of essential oil constituents.
The results demonstrate considerable variation in essential oil yields among accessions.For A. wilhelmsii, the yields ranged from 0.01 to 0.107% in our study, whereas, the literature reports of 0.14-0.82% 27,40,41.Similarly, Rabbi Angouran 30 observed 0.7% yield in A. vermicularis, comparable to our observed range of 0.075-1.5% across accessions.For A. tenuifolia, Sefidkon et al. 28 reported a range of 0.16-1.59%,encompassing our observed variation between accessions of 0.1-2%.Overall, the literature comparisons validate the substantial intra-specific variability in oil yields observed among our Achillea accessions under uniform cultivation.

Essential oil compounds
A total of 75 compounds were identified in the A. wilhelmsii accessions, as presented in Table 2.Among the identified compounds, camphor was determined to be the predominant constituent in this species.The W5 accession exhibited the highest camphor content (31.48%), whereas the remaining seven accessions displayed varying concentrations of this compound (Fig. S1a).The second most significant compound in this species was 1,8-cineole, with concentrations ranging from 4.31% to 18.82%.The W4 accession exhibited the highest proportion of 1,8-cineole and also displayed another notable compound, anethole, at a concentration of 21.63%.
The A. wilhelmsii accessions contained α-pinene in quantities ranging from 1% to 6.7%.The W8 accession displayed a noteworthy amount of piperitone (13.66%), which was only found in small amounts in the other

First year
Second year  Based on the observed variations in compound number and concentration, it can be inferred that there is considerable phytochemical diversity within this species across different regions of the country.The principal compounds identified in the essential oil of the aerial parts of A. wilhelmsii in this study were camphor, 1,8-cineole, anethole, α-pinene, and phytol.
Previous investigations have reported similar compounds, such as camphor, 1,8-cineole, and α-pinene, as well as different compounds, including carvacrol, linalool, and borneol 26,27,42 .The previous reports and the findings of this research suggested that camphor and 1,8-cineole are the principal constituents of the essential oil in this species.Nonetheless, different studies have reported different major compounds for this plant.These disparities may be attributed to variations in physiology, environment, geography, genetics, and plant material diversity 43 .In addition, Saeidi et al. 27 conducted a study to analyze the essential oil composition of twenty A. wilhelmsii accessions collected from their natural habitats across southwest Iran.The researchers identified several components, including chrysanthenone, trans-carveol, linalool, neoiso-dihydrocarveol acetate, camphor, filifolone, 1,8-cineole, borneol, α-pinene, trans-piperitol, (E)-caryophyllene, (E)-nerolidol, and lavandulyl acetate, which were present abundantly in the essential oil of A. wilhelmsii populations.Many of these components were also detected in the accessions studied in the present research.However, certain components such as neoiso-dihydrocarveol acetate, filifolone, and lavandulyl acetate were exclusively identified in the previous study, whereas components like anethole and phytol were specifically identified in the present study.One possible explanation for these differences is that the accessions in the present study were cultivated in specific locations, while Saeidi et al. 27 collected accessions from their natural habitats.Also, in the present study, a broader range of locations across the country was covered, which may explain some of the differences observed compared to the previous study.
A total of 75 compounds were identified in A. vermicularis, similar to A. wilhelmsii, as presented in Table 3.Among these compounds, the composition of 1,8-cineole was recognized as the most significant in this species.The V2 accession exhibited the highest percentage of 1,8-cineole at 26.22% (Fig. S1b).All accessions, except V7, contained varying percentages of this compound.The second most important composition was camphor, with a range of 0% to 28%, and the highest percentage was found in the V6 accession.This accession also exhibited a prominent compound of this species, levo-carvone, at a concentration of 15.38%.The species displayed δ-terpinene in concentrations ranging from 0 to 10%.Several unique compounds were found in the accessions of this species.For instance, V1 contained 10% pinocarvone compound, V7 contained 17.16% cyclohexadecanolide compound, and V9 contained 40.54% pulegone compound.All accessions demonstrated similar percentages of mehp composition.Based on the observed changes in each composition and their respective ranges, it can be inferred that there is significant phytochemical diversity within this species.In a study by Rabbi-Angourani 30 , the main compositions of the essential oil of A. vermicularis were found to be camphor, bornel acetate, and 1 and 8-cineole.This finding aligns with a report on A. vermicularis growth in Turkey, which identified camphor and 15-hexadecanolide as the major components of the essential oil 44 .Previous studies conducted on A. vermicularis from Iran also reported 1,8-cineole, camphor, and germacrene D as the main components 45,46 .In another study by Rezaei et al. 47 , the major constituents of the essential oil were identified as camphor, borneol, and terpinen-4-ol.In the present study, the most important components identified in the essential oil of the aerial parts of A. vermicularis were 1,8-cineole, camphor, δ-terpinene, anethole, borneol, and trans-pinocarveol.These findings are consistent with previous studies conducted in Turkey, which also reported camphor and 1,8-cineole as the most important compounds in this species 48 .
The confirmation of these findings across different regions and years suggests that these compounds are consistently produced under various environmental conditions, although the reported percentages may vary.
The essential oil of A. tenuifolia species yielded a total of 49 identified compounds, as outlined in Table 4.It is worth noting that the number of compounds obtained in A. tenuifolia was significantly lower compared to the other two species investigated.The most prominent compound observed in A. tenuifolia was β-cubebene, which was present in all accessions.Among the accessions, T4 exhibited the highest percentage (50.23%) of β-cubebene, while T3 had the lowest percentage (29.79%)(Fig. S1c).Another notable compound in A. tenuifolia was elixene, with a composition range of 5.95 to 8.91%.The T6 accession displayed the highest percentage (13.88%) of elixene, along with β-sesquiphellandrene. Additionally, two compounds, 1,8-cineole and camphor, were identified as major compounds in A. tenuifolia.These compounds were also found in the other two species, A. vermicularis and A. wilhelmsii, where they constituted the primary compounds.In contrast to the other two species, A. tenuifolia accessions did not exhibit a unique compound, which could be attributed to the close proximity of the sample collection sites or a lower diversity of chemical compounds in this species.Notably, all accessions of A. tenuifolia displayed similar percentages of mehp composition.The key essential compounds identified in the aerial parts of A. tenuifolia in this study were β-cubebene, elixene, β-sesquiphellandrene, 1,8-cineole, camphor, and δ-terpinene.
Previous studies have shed light on the significant chemical compounds of this plant.A study conducted on different parts of the plant reported that flower compounds included limonene and α-cadinol, leaf compounds included limonene, α-pinene, caryophyllene oxide, α-gurjunene, bornyl acetate, and δ-cadinene, while stem compounds included limonene, α-pinene, and spathulenol 49 .Aghjani et al. 50identified camphor and borneol as the primary chemical compounds in the flowers of this plant.The compounds contribute to the diverse biological activities of the essential oil and methanol extract of Achillea species, including antioxidant and antimicrobial properties 44 .
The major components in the essential oil of the three studied Achillea species are presented in Table 5.Among the species, A. tenuifolia exhibited significantly higher amounts of β-cubebene and elixene compared to the other two species.Interestingly, all three species had similar levels of α-pinene.In terms of specific compounds,  2-4).The results showed significant variations in the composition of essential oils among the different Achillea species and their classes.In A. wilhelmsii, the oxygenated monoterpenes are the dominant class, ranging from 35.66 to 61.03% across the eight accessions.The monoterpene hydrocarbons and sesquiterpenes are also present in notable amounts, but in lower proportions compared to the oxygenated monoterpenes.The A. vermicularis samples exhibit a more diverse essential oil profile.The oxygenated monoterpenes are still a significant component, ranging from 14.17 to 71.8%.In contrast, the essential oil composition of A. tenuifolia was dominated by sesquiterpenes, which account for 45.19-70.07% of the total essential oil components across the seven accessions.

Multivariate analysis
Cluster analysis was conducted using the key components of essential oils from the studied accessions, namely β-cubebene, elixene, borneol, camphor, 1,8-cineole, α-pinene, δ-terpinene, phytol, anethole, β-sesquiphellandrene, trans-pinocarveol, and trans-nerolidol.The analysis resulted in the classification of the accessions into three main groups (Fig. 2).The first group comprised seven accessions of A. tenuifolia.Based on the results, the accessions of this species were distinguished from other accessions primarily due to significantly higher levels of β-cubebene and elixene components in their essential oils.Within this group, accession T6 was separated from other A. tenuifolia accessions due to its high level of β-sesquiphellandrene in its essential oil.The second and third groups were formed by the accessions of A. wilhelmsii and A. vermicularis.The cluster analysis did not differentiate between the accessions of these two species, indicating a similarity in their essential oil compositions.However, accession V3 did not belong to the second and third groups due to its elevated levels of anethole and trans-pinocarveol components in its essential oil.The results of principal component analysis (PCA) revealed that the first three principal components (PCs) accounted for 62.21% of the total variance (Table 6).PC1, which explained 28.78% of the variance, exhibited a significant positive correlation with β-cubebene, elixene, and β-sesquiphellandrene, and a significant negative  www.nature.com/scientificreports/correlation with borneol and camphor.PC2, explaining approximately 18.82% of the variance, showed positive correlations with 1,8-cineole, α-pinene, and δ-terpinene, while displaying negative correlations with phytol and anethole.Additionally, PC3 explained 14.61% of the total variation among the study accessions and was positively correlated with trans-pinocarveol, while negatively correlated with trans-nerolidol.The PCA triplot confirmed the clustering results, as the accessions of A. tenuifolia were closely grouped together (Fig. 3).Also, some accessions from A. wilhelmsii and V3 were found to be distant from other accessions of the same species, as well as from A. vermicularis accessions.
Cluster analysis and PCA have played a pivotal role in advancing our understanding of the chemical compositions of essential oils derived from different Achillea species.Yener 51 employed PCA to successfully identify A. nobilis subsp.neilreichii as distinct in terms of its composition, while Turkmenoglu 44 utilized PCA to group species based on their chemotypes.These studies exemplify the effectiveness of PCA in discerning unique chemical profiles within the Achillea genus.Similarly, Sadyrbekov 52 employed cluster analysis to categorize species according to their chemical compositions, further underscoring the significance of these analytical techniques in comprehending the diverse essential oil compositions found in Achillea species.
The correlation coefficients among the top essential oil components of the accessions of three Achillea species, including A. wilhelmsii, A. vermicularis, and A. tenuifolia, were presented as heat map correlation (Fig. 4).The

Antioxidant activity and total phenol content
In this study, the total phenol content and antioxidant activity of the samples were assessed over a period of 2 years.
The analysis of A. wilhelmsii species revealed that the W4 and W7 accessions exhibited higher levels of total phenol content in comparison to other accessions in the initial year (Fig. 5).These two accessions were the sole ones that displayed elevated phenol levels in the first year compared to the second year, while the remaining accessions demonstrated higher phenol levels during the second year relative to the first.A paired t-test was employed to compare the 2 years in terms of this characteristic, which revealed no statistically significant difference between the two periods.Furthermore, the W4 (IC 50 = 278.32)and W7 (IC 50 = 243.21)accessions exhibited greater antioxidant activity in the first year when compared to other samples (Table 7), whereas in the second year, the W3 accession (IC 50 = 203.23)displayed the highest antioxidant activity.With the exception of the W7 accession, all accessions demonstrated higher antioxidant activity in the second year compared to the first.Regression analysis for each accession conducted for each year demonstrated statistically significant models (p < 0.01).Moreover, the coefficient of determination (R-squared) exceeded 0.93 in the majority of models, indicating a high degree of accuracy for the models.
Although there was no significant difference observed in total phenol content across the 2-year period, there was a noteworthy difference in antioxidant activity (p < 0.05).Figure 6 illustrates the relationship between antioxidant activity and total phenol content.Linear regression analysis for these two variables was statistically significant, and the coefficient of determination was relatively high for both years within this species.While phenolic compounds are widely recognized as the principal bioactive compounds associated with antioxidants 16 , it should be noted that total phenol content does not encompass the entirety of antioxidants 53 .
The analysis of A. vermicularis species revealed that the V2 accession exhibited a significantly higher total phenol content in the second year compared to other accessions.Conversely, the V6 accession displayed the highest phenol levels in the first year.Apart from these two accessions, there were no significant differences in phenol content among the accessions over the 2-year period.Notably, the V2 and V6 accessions, characterized by higher phenol levels, also demonstrated superior antioxidant activity compared to other samples.In the second year, all accessions, except for V6, exhibited higher antioxidant activity relative to the first year.The regression analysis results for each accession in each year established the statistical significance of the obtained models (p < 0.01), with the exception of the model associated with the V6 accession in the second year.Furthermore, the coefficient of determination yielded high values in most cases, indicating a strong model accuracy.Although there was no significant variation in total phenol content throughout the 2-year period, antioxidant activity displayed a significant difference (p < 0.01).Linear regression analysis confirmed the significance of this relationship for the two variables in the first year, supporting the linearity of the model.However, in the second year, the relationship between the variables was found to be non-linear.
The analysis of A. tenuifolia species revealed that the highest total phenol contents in the first and second years were obtained from T6 and T3 accessions, respectively.The range of variation in total phenol content was low in the first year but increased in the second year.The t-test analysis indicated a significant difference between the 2 years for both total phenol content and antioxidant activity (p < 0.01).All accessions demonstrated higher antioxidant activity in the second year compared to the first year.The results of regression analysis for each accession in each year revealed the statistical significance of the obtained models (p < 0.01).Additionally, the coefficient of determination exhibited high values in most cases.The results of linear regression analysis revealed a linear relationship between antioxidant activity and total phenol content in both years.
The results demonstrated that among the examined species, A. tenuifolia displayed the highest level of antioxidant activity.However, there was a relatively comparable level of antioxidant activity observed across the studied species.Additionally, A. tenuifolia exhibited the highest total phenol content.
The year factor had a significant effect on the antioxidant activity of all three studied Achillea species, while it was only statistically significant for total phenol content in A. tenuifolia.As the plants are perennial, in the first year of establishment, the growth period until flowering was longer compared to the second year.In the first year, the plants flowered in August, while in the second year flowering occurred in May.The extended growth

Second year
Total phenolic (mg GA/ml) period in the first year likely diverted more resources towards vegetative growth rather than secondary metabolite production 54 .In contrast, the shorter growth cycle in the second year allowed for increased accumulation of phenolic antioxidants in the aerial parts by the time of flowering in May.This may explain the higher antioxidant activity levels observed in all three species during the second year.Meanwhile, the year effect on total phenol content was only significant for A. tenuifolia possibly due to greater sensitivity or capacity for phenolic accumulation in this species.
The results of present study are consistent with a study conducted by Polatoglu et al. 48, which reported significant DPPH scavenging activity in the essential oils of A. tenuifolia and A. vermicularis.Several Achillea species, such as A. vermicularis, A. wilhelmsii, and A. tenuifolia, have been identified as possessing noteworthy antioxidant activity and exhibiting high total phenolic content 45,[55][56][57] .These properties can be attributed to the presence of bioactive compounds, including phenolics and essential oils, in these species 56,58 .However, it should be noted that the antioxidant activity of A. tenuifolia's root extracts does not necessarily correlate with their total phenol content 57 .Also, Al-Ogaili et al. 59 revealed that Iraqi A. tenuifolia contains high levels of polyphenols, indicating its potential as a source of antioxidants.These findings underscore the potential of Achillea species, including A. vermicularis, A. wilhelmsii, and A. tenuifolia, as natural sources of antioxidants with promising applications in the pharmaceutical and medical fields.Also, significant antioxidant properties and total phenol content were observed in Achillea species collected from their original site 56 .These plants exhibited higher antioxidant properties and total phenol content compared to our study, which may be attributed to different factors such as elevation, region, and organs used [60][61][62] .

Conclusion
To highlight the novel findings of this study, the results revealed substantial differences in the essential oil profiles and antioxidant potentials among accessions from three Achillea species (A.wilhelmsii, A. tenuifolia, and A. vermicularis) when cultivated under uniform field conditions.Notably, evaluating multiple accessions together for the first time demonstrated considerable intraspecific chemical diversity and phenolic variations between genotypes of the three species that had not been previously reported.The dominant compounds differed between the species, with camphor being predominant in A. wilhelmsii, 1,8-cineole in A. vermicularis, and β-cubebene and elixene in A. tenuifolia.However, certain compounds, such as 1,8-cineole and camphor, were consistently found across all species.Cluster analysis grouped the accessions into three main clusters, with A. tenuifolia accessions forming a distinct group characterized by higher levels of β-cubebene and elixene.Additionally, the www.nature.com/scientificreports/study assessed the total phenolic content and antioxidant activity of Achillea species over a 2-year period.Among the examined species, A. tenuifolia exhibited the highest levels of total phenol content and antioxidant activity.However, there was a relatively comparable level of antioxidant activity observed across the studied species.Furthermore, linear regression analysis revealed a positive relationship between antioxidant activity and total phenol content in both years for A. tenuifolia and A. wilhelmsii.These findings emphasize the phytochemical diversity within Achillea species and highlight the influence of genetic and environmental factors on their essential oil compositions and antioxidant properties.Moreover, the study underscores the potential of Achillea species as a reliable source of antioxidants for use in the food and pharmaceutical industries.Further research, including the selection of specific accessions within each species, can provide deeper insights into their chemical composition and medicinal potential.

Figure 1 .
Figure 1.Essential oil yield of 25 accessions of the three studied Achillea sp.

Figure 2 .
Figure 2. Heat map clustering of the 25 accessions of three studied Achillea sp based on the eight measured minerals.The color scales represent the values were normalized by Z-score ((value-mean value)/standard error) for each character.

Figure 3 .
Figure 3. PCA triplot based on the three first PC of the 25 accessions of three studied Achillea sp.

Figure 4 .
Figure 4. Heat map correlation among the major essential oil composition of 25 accessions of three studied Achillea sp.

Figure 5 .
Figure 5.Total phenol contents of 25 accessions of the three studied Achillea sp.

Figure 6 .
Figure 6.The relationships between antioxidant activity and total phenol content of three Achillea species over 2 years.

Table 1 .
Geographical location of 25 Iranian Achillea sp accessions.

Table 3 .
Chemical .wilhelmsii displayed higher values of camphor, anethole, phytol, and trans-nerolidol compared to the other two species.On the other hand, the amount of 1,8-cineole in A. vermicularis was approximately double that of A. wilhelmsii, and the amount in A. wilhelmsii was approximately double that of A. tenuifolia.The essential oil components present in four classes (Tables composition of essential oils (%) of ten A. vermicularis accessions.The values in the table are percentages of a given constituent in the total oil.The data were sorted based on the retention index (RI) of the components.Table 4.Chemical composition of essential oils (%) of seven A. tenuifolia accessions.The values in the table are percentages of a given constituent in the total oil.The data were sorted based on the retention index (RI) of the components.Vol.:(0123456789) Scientific Reports | (2024) 14:11843 | https://doi.org/10.1038/s41598-024-62834-1www.nature.com/scientificreports/A

Table 5 .
Mean values of the major essential oil components in the three Achillea sp.

Table 6 .
PCA based on the eight minerals of 25 Achillea sp.accessions.